Tweak receptor

ABSTRACT

The present invention provides the TWEAK receptor and methods for identifying and using agonists and antagonists of the TWEAK receptor. In particular, the invention provides methods of screening for agonists and antagonists and for treating diseases or conditions mediated by angiogenesis, such as solid tumors and vascular deficiencies of cardiac or peripheral tissue.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/754,847, filed Jan. 8, 2004, now allowed, which is a divisional ofU.S. patent application Ser. No. 09/883,777, filed Jun. 18, 2001, nowU.S. Pat. No. 6,727,225, which is a continuation-in-part ofInternational Application Number PCT/US00/34755, filed 19 Dec. 2000, andis a continuation-in-part of U.S. patent application Ser. No.09/742,454, filed Dec. 19, 2000, both of which claim the benefit of U.S.Provisional Application Ser. No. 60/172,878, filed 20 Dec. 1999, andU.S. Provisional Application Ser. No. 60/203,347, filed 10 May 2000. Theabove-identified applications are incorporated herein by reference.

BACKGROUND

Angiogenesis is a multi-step developmental process that results in theformation of new blood vessels off of existing vessels. This spatiallyand temporally regulated process involves loosening of matrix contactsand support cell interactions in the existing vessels by proteases,followed by coordinated movement, morphological alteration, andproliferation of the smooth muscle and endothelial cells of the existingvessel. The nascent cells then extend into the target tissue followed bycell-cell interactions in which the endothelial cells form tubes whichthe smooth muscle cells surround. In a coordinated fashion,extracellular matrix proteins of the vessel are secreted andperi-endothelial support cells are recruited to support and maintainstructural integrity (see, e.g., Daniel et al., Ann. Rev. Physiol.2000(62):649, 2000). Angiogenesis plays important roles in both normaland pathological physiology.

Under normal physiological conditions, angiogenesis is involved in fetaland embryonic development, wound healing, organ regeneration, and femalereproductive remodeling processes including formation of theendometrium, corpus luteum, and placenta. Angiogenesis is stringentlyregulated under normal conditions, especially in adult animals, andperturbation of the regulatory controls can lead to pathologicalangiogenesis.

Pathological angiogenesis has been implicated in the manifestationand/or progression of inflammatory diseases, certain eye disorders, andcancer. In particular, several lines of evidence support the conceptthat angiogenesis is essential for the growth and persistence of solidtumors and their metastases (see, e.g., Folkman, N. Engl. J. Med.285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesisinhibitors are therefore useful for the prevention (e.g., treatment ofpremalignant conditions), intervention (e.g., treatment of smalltumors), and regression (e.g., treatment of large tumors) of cancers(see, e.g. Bergers et al., Science 284:808, 1999).

The TWEAK protein, which has also been called TREPA and Apo3L, is amember of the tumor necrosis factor (TNF) family and is expressed in awide variety of human tissues (Chicheportiche et al., J. Biol. Chem.,272(51):32401, 1997; see also Wiley, PCT Publication No. WO 98/35061, 13Aug. 1998). Like most TNF family members, TWEAK is a Type II membraneprotein with an extracellular C-terminal domain. Although TWEAK wasoriginally described as a weak inducer of apoptosis, this induction ofcell death was later shown to be indirect (Schneider et al., Eur. J.Immunol. 29:1785, 1999).

Lynch et al. demonstrated that TWEAK directly induces endothelial cellproliferation and angiogenesis (J. Biol. Chem., 274(13):8455, 1999).Picomolar concentrations of recombinant soluble TWEAK induceproliferation in multiple endothelial cell lines and in aortic smoothmuscle cells, and reduce the requirement for serum and growth factors inculture. Moreover, TWEAK induces a strong angiogenic response in a ratcorneal pocket assay. Since TNF family members initiate biologicalresponses by signaling through members of the TNF receptor family, therehas been great interest in identifying and characterizing a TWEAKR.

Marsters et al. reported that TWEAK binds to and signals through adeath-domain containing receptor known variously as DR3, Apo3, WSL-1,TRAMP, or LARD (Marsters et al., Current Biology 8(9):525, 1998).Schneider et al., however, showed that TWEAK binds to and signals inKym-1 cells but that Kym-1 cells do not express the receptor DR3(Schneider et al., Eur. J. Immunol. 29:1785, 1999). These resultssuggest the existence of a yet to be identified TWEAK receptor.

Because TWEAK induces angiogenesis in vivo, there is a particular needto identify the major functional TWEAKR. Once identified, TWEAKR may beused to screen for and develop TWEAKR agonists and antagonists for themodulation of angiogenesis and the treatment of human disease.

There is a need for additional compositions and methods of modulatingangiogenesis for the prevention, abrogation, and mitigation of disease.

SUMMARY OF THE INVENTION

The present invention is based upon the identification and biologicalcharacterization of the major functional TWEAK receptor (TWEAKR). Asdescribed below, cDNA encoding the TWEAKR was molecularly cloned from ahuman endothelial cell expression library.

Although DNA and deduced amino acid sequences corresponding to theTWEAKR identified herein have been reported (see, e.g. Kato et al., PCTPublication No. WO 98/55508, 10 Dec. 1998 and Incyte, PCT PublicationNo. WO 99/61471, 2 Dec. 1999), it was not heretofore appreciated thatthese sequences encode a receptor for TWEAK or that the encodedpolypeptide, fragments, agonists, or antagonists thereof can be used tomodulating angiogenesis. Similarly, investigators have recently claimedmethods of making and using TWEAKR antagonists to treat immunologicaldisorders, but without identifying the major TWEAKR or its role inangiogenesis (Rennert, PCT Publication No. WO 00/42073, 20 Jul. 2000).These deficiencies have been addressed, as described herein, byidentification of the major TWEAKR and characterization of itsbiological activities. The identification of TWEAKR has led to thedevelopment of compositions for the modulation of angiogenesis, and alsoprovides screening tools for the identification of diagnostics andtherapeutics.

The invention provides a method of modulating angiogenesis in a subjectin need of such treatment comprising administering atherapeutically-effective amount of a composition comprising a TWEAKR, aTWEAKR antagonist, or a TWEAKR agonist. The subject is a mammal,preferably a human. A composition useful in the methods of the inventioncomprises a TWEAKR polypeptide having a sequence as set forth in SEQ IDNO:4, including fragments and variants thereof. In one embodiment, thecomposition comprises a sequence of amino acids from about residue 28 toan amino acid residue from about 68-80 of SEQ ID NO:4. In one embodimentangiogenesis is inhibited. A TWEAKR antagonist includes, for example, asoluble TWEAKR fragment; an anti-TWEAKR antibody; an antisense or triplehelix forming nucleic acid that binds to a TWEAK or TWEAKR nucleic acidmolecule; peptides; and small molecules. In another embodiment, theTWEAKR antagonist comprises the extracellular domain of TWEAKR, orfragment thereof, and an Fc or leucine zipper domain.

The invention also provides a method of modulating the interaction ofTWEAK and TWEAKR. The method includes contacting a cell comprising TWEAKor TWEAKR with a composition comprising an agent selected from the groupconsisting of: (a) a soluble TWEAKR extracellular domain, and (b) anantibody that binds to the TWEAKR extracellular domain, under conditionssuch that the cell and the composition interact. The contacting may bein vitro, ex vivo, or in vivo.

The invention further provides a method for targeting a detectable labelor chemotherapeutic to a vascular tissue comprising contacting thevascular tissue with an antibody that binds TWEAKR.

In yet another aspect the invention provides a purified polypeptideconsisting of amino acid 28 to x₁ of SEQ ID NO:4, wherein x₁ is an aminoacid from about 68 to 80 of SEQ ID NO:4 and naturally occurring variantsthereof.

Also provided by the invention is a fusion polypeptide comprising apolypeptide consisting of amino acid 28 to x₁ of SEQ ID NO:4, wherein x₁is an amino acid from about 68 to 80 of SEQ ID NO:4 and naturallyoccurring variants thereof, operably linked to a polypeptide ofinterest. The polypeptide of interest can be any series of amino acidsand includes an Fc polypeptide, leucine zipper polypeptide, or anpeptide linker. In one embodiment the fusion polypeptide has a sequenceas set forth in SEQ ID NO: 7 or 9.

The invention also provides an isolated polypeptide comprising asequence Z₁-X-Z₂, wherein Z₁ and Z₂ are each individually a polypeptideconsisting of amino acid 28 to x₁ of SEQ ID NO:4, wherein x₁ is an aminoacid from about 68 to 80 of SEQ ID NO:4 and naturally occurring variantsthereof, and X is a peptide linker. In one embodiment Z₁ and Z₂ have thesame amino acid sequence. In another embodiment Z₁ and Z₂ have differentamino acid sequences. The X moiety can be any peptide linker including,for example, --Gly-Gly--, GGGGS (SEQ ID NO:10) (GGGGS)_(n) (SEQ IDNO:11), GKSSGSGSESKS (SEQ ID NO:12), GSTSGSGKSSEGKG (SEQ ID NO:13),GSTSGSGKSSEGSGSTKG (SEQ ID NO:14), GSTSGSGKPGSGEGSTKG (SEQ ID NO:15),and EGKSSGSGSESKEF (SEQ ID NO:16). In another embodiment, thepolypeptide comprises Z₁-X-Z₂(—X-Z)_(n), wherein ‘n’ is any integer, butis preferably 1 or 2.

The invention further provides an isolated polynucleotide encoding thepurified polypeptide consisting of amino acid 28 to x₁ of SEQ ID NO:4,wherein x₁ is an amino acid from about 68 to 80 of SEQ ID NO:4 andnaturally occurring variants thereof. In one embodiment, thepolynucleotide as a sequence of nucleotides from about 134 to x₂ of SEQID NO:3, wherein x₂ is a nucleotide from about 256 to 292 of SEQ IDNO:3.

The invention provides an isolated polynucleotide encoding a fusionpolypeptide of the invention. In one embodiment, the polynucleotide hasa sequence of nucleotides from about 134 to x₂ of SEQ ID NO:3, whereinx₂ is a nucleotide from about 256 to 292 of SEQ ID NO:3. In anotheraspect, the polynucleotide has a sequence as set forth in SEQ ID NO:6 or8.

The invention includes vectors comprising the polynucleotide of theinvention as well as host cells comprising a polynucleotide of theinvention. The host cell can be an animal cell, a plant cell, and abacterial cell (e.g., E. coli, Bacillus subtilis, and Salmonellatyphimurium).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a sequence alignment of the human and murine TWEAKRpolypeptide sequences. The top sequence is the murine TWEAKR polypeptide(SEQ ID NO:5), and the bottom sequence is the human TWEAKR polypeptide(SEQ ID NO:4).

FIG. 1B shows the primary amino acid sequence of TweakR showing majorfeatures. The leader sequence in underlined. The arrow indicates thepredicted site of cleavage of the leader sequence. The region of TNFfamily receptor homology is shown in bold. The predicted transmembraneregion is doubly underlined. The putative c TRAF binding motif in thecytoplasmic domain is boxed.

FIG. 2 shows the effect of TWEAKR-Fc on PMA-induced HRMEC wound closure.

FIG. 3 shows the effect of TWEAKR-Fc on EGF-induced HRMEC wound closure.

FIG. 4 shows the effect of human TWEAKR-Fc on TWEAK-induced (100 ng/ml)HUVEC proliferation.

FIG. 5 shows the effect of human TWEAKR-Fc on FGF-2-induced (10 ng/ml)HUVEC proliferation.

FIG. 6 collectively shows a scatchard analysis of TWEAK-TWEAKRinteraction. CV-1 cells transfected with human full-length TWEAK mixed1:30 with Raji cells and incubated with various concentrations of¹²⁵I-labeled TWEAKR-Fc. A) Shows scatchard representation of specificbinding. B) Plot of competitive inhibition of unlabeled vs. ¹²⁵I-labeledTWEAKR-Fc.

FIG. 7 collectively shows that human TWEAKR-Fc inhibits PMA- orEGF-stimulated endothelial cell migration in vitro. A, Shows thatTWEAKR-Fc inhibited the PMA-stimulated migration rate to baseline atconcentrations greater than or equal to 1.5 μg/ml, whereas huIgG atsimilar concentrations did not effect migration. Neither huIgG norTweakR-Fc increased or decreased the basal migration rate when added tothe cultures alone. B, Human TweakR-Fc inhibits EGF-induced endothelialcell migration. TweakR-Fc inhibited EGF-stimulated migration to basallevels at 5 μg/ml. Partial inhibition of EGF-induced migration was alsoobserved at huTweakR/Fc concentrations of 500 ng/ml and 1.5 μg/ml.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to TWEAKR and methods for identifyingand using agonists and antagonists of TWEAKR. The invention providesmethods of screening for agonists and antagonists and for treatingdiseases or conditions mediated by angiogenesis.

Abbreviations and Terminology Used in the Specification

“4-1BB” and “4-1BB ligand” (4-1BB-L) are polypeptides described, interalia, in U.S. Pat. No. 5,674,704, including soluble forms thereof.

“CD40 ligand” (CD40L) is a polypeptide described, inter alia, in U.S.Pat. No. 5,716,805, including soluble forms thereof.

“Flt3L” is Flt3 ligand, a polypeptide described, inter alia, in U.S.Pat. No. 5,554,512, including soluble forms thereof.

“RTKs” are receptor tyrosine kinases.

“Tek,” which has also been called Tie2 and ork, is an RTK that ispredominantly expressed in vascular endothelium. The molecular cloningof human Tek (ork) has been described by Ziegler, U.S. Pat. No.5,447,860. “Tek antagonists” are described, inter alia, in Cerretti etal., PCT Publication No. WO 00/75323, 14 Dec. 2000.

“TRAIL” is TNF-related apoptosis-inducing ligand, a type IItransmembrane polypeptide in the TNF family described, inter alia, inU.S. Pat. No. 5,763,223, including soluble forms thereof.

“VEGF” is vascular endothelial growth factor, also known as VPF orvascular permeability factor.

Soluble TWEAKR Polypeptides

As described in the examples below, the native human TWEAKR cDNA has asequence as set forth in SEQ ID NO:3, which encodes a 129 residuepolypeptide (SEQ ID NO:4). Several distinct regions can be discernedwithin the TWEAKR polypeptides of the invention (see, e.g., FIG. 1). Aleader sequence, also called a signal peptide, is present in thesepolypeptides. The leader sequence present in the full-length TWEAKRpolypeptide of the invention is predicted to include amino acids 1-27 ofSEQ ID NO:4. The signal peptide cleavage site for TWEAKR polypeptide canbe predicted using a computer algorithm. However, one of skill in theart will recognize that the cleavage site of the signal sequence mayvary depending upon a number of factors including the organism in whichthe polypeptide is expressed. Accordingly, the N-terminus of a matureform of a TWEAKR polypeptide of the invention may vary by about 2 to 5amino acids. Thus, a mature form of a TWEAKR polypeptide of theinvention may include at its N-terminus amino acid 22, 23, 24, 25, 26,27, 28, 29, 30, 31, or 32 of SEQ ID NO:4. Accordingly, a mature form ofa TWEAKR polypeptide includes amino acids 23, 24, 25, 26, 27, 28, 29,30, 31, 32 or 33 to about amino acid 129 (or, in the case of a solublepolypeptide, an amino acid between 68 and 80) of SEQ ID NO:4. Theextracellular region of a TWEAKR polypeptide is located at about aminoacids 28 to 80 of SEQ ID NO:4. The transmembrane region for the TWEAKRpolypeptide is located at about amino acids 81 to 100 of SEQ ID NO:4.The intracellular region is located at about amino acids 101 to 129 ofSEQ ID NO:4. A putative TWEAKR sequence has also been reported by Katoet al., PCT Publication No. WO 98/55508, 10 Dec. 1998 and by Incyte, PCTPublication No. WO 99/61471, 2 Dec. 1999. As used herein, “TWEAKR”includes polypeptides having these sequences, and in particularcomprising amino acids 28 to x₁ of SEQ ID NO:4, wherein x₁ is an aminoacid from 68 to 80 of SEQ ID NO:4, as well as naturally occurringvariants thereof.

The invention provides both full-length and mature forms of TWEAKRpolypeptides. Full-length polypeptides are those having the completeprimary amino acid sequence of the polypeptide as initially translated(see, e.g., SEQ ID NO:4). The amino acid sequences of full-lengthpolypeptides can be obtained, for example, by translation of thecomplete open reading frame (“ORF”) of a cDNA molecule (see, e.g. SEQ IDNO:3). An example of a full length TWEAKR polypeptide of the inventioncomprises a sequence as set forth in SEQ ID NO:4 from amino acid 1 toamino acid 129. Such a full length polypeptide is contemplated toinclude, for example, the signal peptide comprising amino acid 1 toabout amino acid 27 of SEQ ID NO:4.

The “mature form” of a polypeptide refers to a polypeptide that hasundergone post-translational processing steps, if any, such as, forexample, cleavage of the signal sequence or proteolytic cleavage toremove a prodomain. Multiple mature forms of a particular full-lengthpolypeptide may be produced, for example, by imprecise cleavage of thesignal sequence, or by differential regulation of proteases that cleavethe polypeptide. The mature form(s) of such polypeptide may be obtainedby expression, in a suitable mammalian cell or other host cell, of apolynucleotide that encodes the full-length polypeptide. The sequence ofthe mature form of the polypeptide may also be determinable from theamino acid sequence of the full-length form, through identification ofsignal sequences or protease cleavage sites (e.g., a protease cleavagesite is predicted between the Gly-Glu residues at positions 27 and 28 ofSEQ ID NO:4). An example of a mature form of a TWEAKR polypeptide of theinvention comprises a sequence as set forth in SEQ ID NO:4 from aboutamino acid 28 to amino acid 129.

In another aspect of the invention, fragments of TWEAKR polypeptides areprovided. Such fragments include, for example, the various domainsidentified above (e.g. the signal sequence domain, the extracellulardomain, the transmembrane domain, and the cytoplasmic or intracellulardomain). Such domains find use in recombinant DNA techniques (e.g.,creation of fusion proteins and the like). Of particular interest is theextracellular domain of TWEAKR from about amino acid 28 to amino acid 68to 80 of SEQ ID NO:4. The extracellular domain of TWEAKR comprises asoluble TWEAKR amino acid sequence. Also included in the invention arefragments of the extracellular domain that retain a biological activityof TWEAKR. For example, a biological activity associated with a TWEAKRextracellular domain or fragment thereof includes the ability to bind toTWEAK.

In one aspect of the invention, a soluble TWEAKR fragment is used as aTWEAKR antagonist to inhibit angiogenesis and/or to inhibit the bindingof TWEAK ligand to TWEAKR. A TWEAKR fragment preferably comprises theextracellular domain of TWEAKR or a portion thereof as described hereinsuch that the fragment comprises a soluble TWEAKR amino acid sequence.Accordingly, a TWEAKR antagonist includes, for example, a solubleportion of the TWEAKR molecule, preferably a portion of theextracellular domain of TWEAKR, either alone, fused, or conjugated toone or more other molecules or polypeptides (e.g., an Fc, leucine zipperpolypeptide, or a peptide linker). For example, the invention providescompositions and fusion proteins that comprise at least one solubleTWEAKR polypeptide domain (e.g., the extracellular domain).

Soluble polypeptides are capable of being secreted from the cells inwhich they are expressed. The use of soluble forms of polypeptides isadvantageous for certain applications. Purification of the polypeptidesfrom recombinant host cells is facilitated since the polypeptides aresecreted, and soluble proteins are generally suited for parenteraladministration. A secreted soluble polypeptide may be identified (anddistinguished from its non-soluble membrane-bound counterparts) byseparating intact cells which express the desired polypeptide from theculture medium, e.g. by centrifugation, and assaying the medium(supernatant) for the presence of the desired polypeptide. The presenceof the desired polypeptide in the medium indicates that the polypeptidewas secreted from the cells and thus is a soluble form of thepolypeptide. Soluble polypeptides may be prepared by any of a number ofconventional techniques. A polynucleotide encoding a desired solublepolypeptide may be subcloned into an expression vector for production ofthe polypeptide, or the desired encoding polynucleotide or solublepolypeptide may be chemically synthesized. Examples of a nucleic acidmolecule encoding a soluble TWEAKR polypeptide comprises aboutnucleotides 134 to 256, 134 to 262, 134 to 289, and 134 to 292 of SEQ IDNO:3. In one embodiment, D-amino acids are substituted for the naturallyoccurring L-amino acids. D-amino acids provide improved stability underin vivo conditions. In addition, due to the size of the extracellulardomain or soluble polypeptide sequence of the invention it may beadvantageous to synthesize the polypeptide using D-amino acids. It willbe recognized that the polypeptide of the invention can be synthesizedsuch that the polypeptide comprises a combination of L- and D-aminoacids.

Soluble TWEAKR polypeptides comprise all or part of the TWEAKRextracellular domain, but generally lack the transmembrane domain thatwould cause retention of the polypeptide at the cell surface. Solublepolypeptides may include part of the transmembrane domain or all or partof the cytoplasmic domain so long as the polypeptide is secreted fromthe cell in which it is produced. Soluble TWEAKR polypeptidesadvantageously comprise a native or heterologous signal peptide wheninitially synthesized, to promote secretion from the cell, but thesignal sequence is cleaved upon secretion. The term “TWEAKRextracellular domain” is intended to encompass all or part of the nativeTWEAKR extracellular domain, as well as related forms including but notlimited to: (a) fragments, (b) variants, (c) derivatives, and (d) fusionpolypeptides. The ability of these related forms to inhibit angiogenesisor other TWEAKR-mediated responses may be determined in vitro or invivo, using methods such as those exemplified below or using otherassays known in the art. Examples of soluble TWEAKR polypeptides areprovided below. In some embodiments of the present invention amultimeric form of a soluble TWEAKR polypeptide (“soluble TWEAKRmultimer”) is used as an antagonist to block the binding of TWEAK toTWEAKR, to inhibit angiogenesis or other TWEAKR-mediated responses.

Soluble TWEAKR multimers are covalently-linked or non-covalently-linkedmultimers, including dimers, trimers, or higher multimers. Multimers maybe linked by disulfide bonds formed between cysteine residues ondifferent soluble TWEAKR polypeptides. One embodiment of the inventionis directed to multimers comprising multiple soluble TWEAKR polypeptidesjoined via covalent or non-covalent interactions between peptidemoieties fused to the soluble TWEAKR polypeptides. Such peptides may bepeptide linkers (spacers), or peptides that have the property ofpromoting multimerization. In one embodiment peptide linkers are fusedto the C-terminal end of a first soluble TWEAKR molecule and theN-terminal end of a second soluble TWEAKR molecule. This structure maybe repeated multiple times such that at least one, preferably 2, 3, 4,or more soluble TWEAKR polypeptides are linked to one another viapeptide linkers at their respective termini. For example, a polypeptideof the invention comprises a sequence Z₁-X-Z₂, wherein Z₁ and Z₂ areeach individually a polypeptide consisting of amino acid 28 to x₁ of SEQID NO:4, wherein x₁ is an amino acid from about 68 to 80 of SEQ ID NO:4and X is a peptide linker. In another embodiment, the polypeptidecomprises Z₁-X-Z₂(—X-Z)_(n), wherein ‘n’ is any integer, but ispreferably 1 or 2. In a further embodiment, the peptide linkers shouldbe of sufficient length to allow the soluble TWEAKR polypeptide to formbonds with adjacent soluble TWEAKR polypeptides. Examples of peptidelinkers include --Gly-Gly--, GGGGS (SEQ ID NO: 10) (GGGGS)_(n) (SEQ IDNO:11), GKSSGSGSESKS (SEQ ID NO: 12), GSTSGSGKSSEGKG (SEQ ID NO:13),GSTSGSGKSSEGSGSTKG (SEQ ID NO:14), GSTSGSGKPGSGEGSTKG (SEQ ID NO:15), orEGKSSGSGSESKEF (SEQ ID NO:16). Linking moieties are described, forexample, in Huston, J. S., et al., PNAS 85:5879-5883 (1988), Whitlow,M., et al., Protein Engineering 6:989-995 (1993), and Newton, D. L., etal., Biochemistry 35:545-553 (1996). Other suitable peptide linkers arethose described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which arehereby incorporated by reference. A polynucleotide encoding a desiredpeptide linker can be inserted between, and in the same reading frameas, a polynucleotide encoding a soluble TWEAKR polypeptide, using anysuitable conventional technique. In particular embodiments, a fusionpolypeptide comprises from two to four soluble TWEAKR polypeptidesseparated by peptide linkers.

In some embodiments, a soluble TWEAKR multimer is prepared usingpolypeptides derived from immunoglobulins. Preparation of fusionproteins comprising certain heterologous polypeptides fused to variousportions of antibody-derived polypeptides (including the Fc domain) hasbeen described, e.g., by Ashkenazi et al. (Proc. Natl. Acad. Sci. USA88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).

One preferred embodiment of the present invention is directed to aTWEAKR-Fc dimer comprising two fusion proteins created by fusing asoluble TWEAKR to an Fc polypeptide. A gene fusion encoding theTWEAKR-Fc fusion protein is inserted into an appropriate expressionvector. TWEAKR-Fc fusion proteins are expressed in host cellstransformed with the recombinant expression vector, and allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between the Fc moieties to yield divalent soluble TWEAKR. Theterm “Fc polypeptide” as used herein includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization are also included.

One suitable Fc polypeptide, described in PCT application WO 93/10151,is a single chain polypeptide extending from the N-terminal hinge regionto the native C-terminus of the Fc region of a human IgG1 antibody.Another useful Fc polypeptide is the Fc mutein described in U.S. Pat.No. 5,457,035 and by Baum et al., EMBO J. 13:3992, 1994. The amino acidsequence of this mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors. Fusion polypeptides comprising Fcmoieties, and multimers formed therefrom, offer an advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns, and Fc fusion polypeptides may provide a longer in vivo halflife, which is useful in therapeutic applications, than unmodifiedpolypeptides.

In other embodiments, a soluble TWEAKR polypeptide may be substitutedfor the variable portion of an antibody heavy or light chain. If fusionproteins are made with both heavy and light chains of an antibody, it ispossible to form a soluble TWEAKR multimer with as many as four solubleTWEAKR polypeptides.

Another method for preparing soluble TWEAKR multimers involves use of aleucine zipper domain. Leucine zipper domains are peptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988), and have since been found in a varietyof different proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solublemultimeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al.,Semin. Immunol. 6:267, 1994. Recombinant fusion proteins comprising asoluble TWEAKR polypeptide fused to a leucine zipper peptide areexpressed in suitable host cells, and the soluble TWEAKR multimer thatforms is recovered from the culture supernatant.

For some applications, the soluble TWEAKR multimers of the presentinvention are believed to provide certain advantages over the use ofmonomeric forms. Fc fusion polypeptides, for example, typically exhibitan increased in vivo half-life as compared to an unmodified polypeptide.

The present invention encompasses the use of various forms of solubleTWEAKR multimers that retain the ability to inhibit angiogenesis orother TWEAKR-mediated responses. The term “soluble TWEAKR multimer” isintended to encompass multimers containing all or part of the nativeTWEAKR extracellular domain, as well as related forms including, but notlimited to, multimers of: (a) fragments, (b) variants, (c) derivatives,and (d) fusion polypeptides of soluble TWEAKR. The ability of theserelated forms to inhibit angiogenesis or other TWEAKR-mediated responsesmay be determined in vitro or in vivo, using methods such as thoseexemplified in the examples or using other assays known in the art.

Among the soluble TWEAKR polypeptides and soluble TWEAKR multimersuseful in practicing the present invention are TWEAKR variants thatretain the ability to bind ligand (e.g. TWEAK) and/or inhibitangiogenesis or other TWEAKR-mediated responses. Such TWEAKR variantsinclude polypeptides that are substantially homologous to native TWEAKR,but which have an amino acid sequence different from that of a nativeTWEAKR because of one or more deletions, insertions or substitutions.Particular embodiments include, but are not limited to, TWEAKRpolypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a native TWEAKRsequence. Included as variants of TWEAKR polypeptides are those variantsthat are naturally occurring, such as allelic forms and alternativelyspliced forms, as well as variants that have been constructed bymodifying the amino acid sequence of a TWEAKR polypeptide or thenucleotide sequence of a nucleic acid encoding a TWEAKR polypeptide.

Generally, substitutions for one or more amino acids present in thenative polypeptide should be made conservatively. Examples ofconservative substitutions include substitution of amino acids outsideof the active domain(s), and substitution of amino acids that do notalter the secondary and/or tertiary structure of TWEAKR. Additionalexamples include substituting one aliphatic residue for another, such asIle, Val, Leu, or Ala for one another, or substitutions of one polarresidue for another, such as between Lys and Arg; Glu and Asp; or Glnand Asn, or substitutions of one aromatic residue for another, such asPhe, Trp, or Tyr for one another. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are known in the art.

In some preferred embodiments, the TWEAKR variant is at least about 70%identical in amino acid sequence to the amino acid sequence of nativeTWEAKR; in some preferred embodiments, the TWEAKR variant is at leastabout 80% identical in amino acid sequence to the amino acid sequence ofnative TWEAKR. In some more preferred embodiments, the TWEAKR variant isat least about 90% identical in amino acid sequence to the amino acidsequence of native TWEAKR; in some more preferred embodiments, theTWEAKR variant is at least about 95% identical in amino acid sequence tothe amino acid sequence of native TWEAKR. In some most preferredembodiments, the TWEAKR variant is at least about 98% identical in aminoacid sequence to the amino acid sequence of native TWEAKR; in some mostpreferred embodiments, the TWEAKR variant is at least about 99%identical in amino acid sequence to the amino acid sequence of nativeTWEAKR. Percent identity, in the case of both polypeptides and nucleicacids, may be determined by visual inspection. Percent identity may alsobe determined using the alignment method of Needleman and Wunsch (J.Mol. Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl.Math 2:482, 1981. Preferably, percent identity is determined by using acomputer program, for example, the GAP computer program version 10.xavailable from the Genetics Computer Group (GCG; Madison, Wis., see alsoDevereux et al., Nucl. Acids Res. 12:387, 1984). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979 for amino acids; (2) a penalty of30 (amino acids) or 50 (nucleotides) for each gap and an additional 1(amino acids) or 3 (nucleotides) penalty for each symbol in each gap;(3) no penalty for end gaps; and (4) no maximum penalty for long gaps.Other programs used by one skilled in the art of sequence comparison mayalso be used. For fragments of TWEAKR, the percent identity iscalculated based on that portion of TWEAKR that is present in thefragment.

The present invention further encompasses the use of soluble TWEAKRpolypeptides with or without associated native-pattern glycosylation.TWEAKR expressed in yeast or mammalian expression systems (e.g. COS-1 orCOS-7 cells) may be similar to or significantly different from a nativeTWEAKR polypeptide in molecular weight and glycosylation pattern,depending upon the choice of expression system. Expression of TWEAKRpolypeptides in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Different host cells may also processpolypeptides differentially, resulting in heterogeneous mixtures ofpolypeptides with variable N- or C-termini.

The primary amino acid structure of soluble TWEAKR polypeptides may bemodified to create derivatives by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like. Covalent derivatives ofTWEAKR may be prepared by linking particular functional groups to TWEAKRamino acid side chains or at the N-terminus or C-terminus of a TWEAKRpolypeptide. In addition, TWEAKR can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like,or incorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

Fusion polypeptides of soluble TWEAKR that are useful in practicing theinvention also include covalent or aggregative conjugates of a TWEAKRpolypeptide with other polypeptides added to provide novelpolyfunctional entities.

TWEAKR Antibodies

One aspect of the present invention relates to the antigenic epitopes ofthe TWEAKR extracellular domain. Such epitopes are useful for raisingantibodies, and in particular the blocking monoclonal antibodiesdescribed in more detail below. Such epitopes or variants thereof can beproduced using techniques well known in the art such as solid-phasesynthesis, chemical or enzymatic cleavage of a polypeptide, or usingrecombinant DNA technology.

The claimed invention encompasses compositions and uses of antibodiesthat are immunoreactive with TWEAKR polypeptides. Such antibodies “bindspecifically” to TWEAKR polypeptides, meaning that they bind viaantigen-binding sites of the antibody as compared to non-specificbinding interactions. The terms “antibody” and “antibodies” are usedherein in their broadest sense, and include, without limitation, intactmonoclonal and polyclonal antibodies as well as fragments such as Fv,Fab, and F(ab′)2 fragments, single-chain antibodies such as scFv, andvarious chain combinations. The antibodies of the present invention arepreferably humanized, and more preferably human. The antibodies may beprepared using a variety of well-known methods including, withoutlimitation, immunization of animals having native or transgenic immunerepertoires, phage display, hybridoma and recombinant cell culture, andtransgenic plant and animal bioreactors.

Both polyclonal and monoclonal antibodies may be prepared byconventional techniques. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide, harvesting spleen cells from the immunizedanimal, fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells, and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies produced by hybridomas may be recovered byconventional techniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., “humanized” versions of antibodies originally producedin mice or other non-human species. A humanized antibody is anengineered antibody that typically comprises the variable region of anon-human (e.g., murine) antibody, or at least complementaritydetermining regions (CDRs) thereof, and the remaining immunoglobulinportions derived from a human antibody. Procedures for the production ofchimeric and further engineered monoclonal antibodies include thosedescribed in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, May, 1993). Such humanized antibodies may beprepared by known techniques and offer the advantage of reducedimmunogenicity when the antibodies are administered to humans.

Procedures that have been developed for generating human antibodies innon-human animals may be employed in producing antibodies of the presentinvention. The antibodies may be partially human or preferablycompletely human. For example, transgenic mice into which geneticmaterial encoding one or more human immunoglobulin chains has beenintroduced may be employed. Such mice may be genetically altered in avariety of ways. The genetic manipulation may result in humanimmunoglobulin polypeptide chains replacing endogenous immunoglobulinchains in at least some, and preferably virtually all, antibodiesproduced by the animal upon immunization.

Mice in which one or more endogenous immunoglobulin genes have beeninactivated by various means have been prepared and are commerciallyavailable from, for example, Medarex Inc. (Princeton, N.J.) and AbgenixInc. (Fremont, Calif.). Human immunoglobulin genes have been introducedinto the mice to replace the inactivated mouse genes. Antibodiesproduced in the animals incorporate human immunoglobulin polypeptidechains encoded by the human genetic material introduced into the animal.Examples of techniques for the production and use of such transgenicanimals to make antibodies (which are sometimes called “transgenicantibodies”) are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and5,545,806, which are incorporated by reference herein.

Inhibitory Antisense, Ribozyme, and Triple Helix Approaches

Modulation of angiogenesis in a tissue or group of cells may also beameliorated by decreasing the level of TWEAKR gene expression and/orTWEAKR-ligand interaction by using TWEAKR or ligand gene sequences inconjunction with well-known antisense, gene “knock-out,” ribozyme and/ortriple helix methods to decrease the level of TWEAKR or ligand geneexpression. Among the compounds that may exhibit the ability to modulatethe activity, expression or synthesis of the TWEAKR or a ligand gene,including the ability to modulate angiogenesis, are antisense, ribozyme,and triple helix molecules. Such molecules may be designed to reduce orinhibit either unimpaired, or if appropriate, mutant target geneactivity. Techniques for the production and use of such molecules areknown to those of skill in the art.

Recombinant Production of TWEAKR Polypeptides

TWEAKR polypeptides, including soluble TWEAKR polypeptides, fragments,and fusion polypeptides, used in the present invention may be preparedusing a recombinant expression system. Host cells transformed with arecombinant expression vector or a polynucleotide encoding a TWEAKRpolypeptide, soluble TWEAKR polypeptide, or fusion polypeptide(“recombinant host cells”) are cultured under conditions that promoteexpression of TWEAKR molecule and the TWEAKR molecule is recovered.TWEAKR polypeptides can also be produced in transgenic plants oranimals, or by chemical synthesis.

TWEAKR Nucleic Acids

The invention encompasses nucleic acid molecules (i.e., polynucleotides)encoding a TWEAKR polypeptide used in the invention, including: (a)nucleic acids that encode residues from about 28 to x₁ (x₁ is residue68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80) of SEQ ID NO:4 andfragments thereof that bind TWEAK; (b) nucleic acids that are at least70%, 80%, 90%, 95%, 98%, or 99% identical to a nucleic acid of (a), andwhich encode a polypeptide capable of binding TWEAK; and (c) nucleicacids that hybridize at moderate stringency to a nucleic acid of (a),and which encode a polypeptide capable of binding TWEAK.

Due to degeneracy of the genetic code, there can be considerablevariation in nucleotide sequences encoding the same amino acid sequence.Included as embodiments of the invention are nucleic acid sequencescapable of hybridizing under moderately stringent conditions (e.g.prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of 50° C., 5×SSC, overnight) to the DNAsequences encoding TWEAKR. The skilled artisan can determine additionalcombinations of salt and temperature that constitute moderatehybridization stringency (see also, Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1982; and Ausubel, Current Protocols in Molecular Biology, Wileyand Sons, 1989 and later versions, which are incorporated herein byreference). Conditions of higher stringency include higher temperaturesfor hybridization and post-hybridization washes, and/or lower saltconcentration. Percent identity of nucleic acids may be determined usingthe methods described above for polypeptides, e.g., by methods includingvisual inspection and/or the use of computer programs such as GAP.

Any suitable expression system may be employed for the production ofrecombinant TWEAKR. Recombinant expression vectors include nucleic acids(e.g., DNA or RNA) encoding a TWEAKR polypeptide operably linked tosuitable transcriptional and translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. A TWEAKR nucleic acid molecule and a regulatory sequenceare operably linked when the regulatory sequence functionally relates tothe TWEAKR nucleic acid molecule. Thus, a regulatory sequence such as apromoter is operably linked to a TWEAKR nucleic acid molecule if thepromoter controls the transcription of the TWEAKR nucleic acid molecule.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, internalribosome entry sites (IRES), and appropriate sequences which controltranscription and translation initiation and termination. A sequenceencoding an appropriate signal peptide (native or heterologous) can beincorporated into expression vectors. A DNA sequence for a signalpeptide (referred to by a variety of names including secretory leader,leader peptide, or leader) may be fused in frame to the TWEAKR sequenceso that the TWEAKR polypeptide is initially translated as a fusionprotein comprising the signal peptide. A signal peptide that isfunctional in the intended host cells promotes extracellular secretionof the TWEAKR polypeptide. The signal peptide is cleaved from the TWEAKRpolypeptide upon secretion of TWEAKR from the cell.

Suitable host cells for expression of TWEAKR polypeptides includeprokaryotes, yeast, and higher eukaryotic cells, including insect andmammalian cells. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, insect, and mammalian cellular hosts aredescribed, for example, in Pouwels et al. Cloning Vectors: A LaboratoryManual, Elsevier, New York, 1985.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, TWEAKR polypeptides may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker gene(s). A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a TWEAKRDNA sequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

The stability of TWEAKR lends itself to expression in prokaryoticsystems. For example, TweakR ligand binding domain will spontaneouslyre-fold into an active conformation even after being reduced and boiledin SDS loading buffer.

TWEAKR polypeptides may also be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phospho-glucose isomerase, and glucokinase. Other suitablevectors and promoters for use in yeast expression are further describedin Hitzeman, EPA-73,657. Another alternative is the glucose-repressibleADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674,1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectorsreplicable in both yeast and E. coli may be constructed by inserting DNAsequences from pBR322 for selection and replication in E. coli (Amprgene and origin of replication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof recombinant polypeptides. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Insect host cell culture systems also may be employed to expressrecombinant TWEAKR polypeptides, including soluble TWEAKR polypeptides.Bacculovirus systems for production of heterologous polypeptides ininsect cells are reviewed by Luckow and Summers, Bio/Technology 6:47,1988.

Mammalian cells are typically used as host cells. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLacells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell linederived from the African green monkey kidney cell line CV1 (ATCC CCL 70)as described by McMahan et al. (EMBO J. 10: 2821, 1991). For theproduction of therapeutic polypeptides it is particularly advantageousto use a mammalian host cell line which has been adapted to grow inmedia that does not contain animal proteins.

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used totransfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413,1987). In addition, electroporation can be used to transfect mammaliancells using conventional procedures, such as those in Sambrook et al.Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold SpringHarbor Laboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216, 1980). A plasmidexpressing the DHFR cDNA can be introduced into strain DX-B11, and onlycells that contain the plasmid can grow in the appropriate selectivemedia. Other examples of selectable markers that can be incorporatedinto an expression vector include cDNAs conferring resistance toantibiotics, such as G418 and hygromycin B. Cells harboring the vectorcan be selected on the basis of resistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534) and thetripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras etal., J. Biol. Chem. 257:13475, 1982). The internal ribosome entry site(IRES) sequences of viral origin allows dicistronic mRNAs to betranslated efficiently (Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295, 1993; Ramesh et al., Nucleic Acids Research 24:2697,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150, 1997, and p2A51described by Morris et al., Animal Cell Technology, 1997, pp. 529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335, 1989. Other expression vectors for use in mammalianhost cells can be constructed as disclosed by Okayama and Berg (Mol.Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are known inthe art.

Regarding signal peptides that may be employed in producing TWEAKRpolypeptides, the native TWEAKR signal peptide may used or it may bereplaced by a heterologous signal peptide or leader sequence, ifdesired. The choice of signal peptide or leader may depend on factorssuch as the type of host cells in which the recombinant TWEAKR is to beproduced. Examples of heterologous signal peptides that are functionalin mammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

Using the techniques of recombinant DNA including mutagenesis, directedevolution, and the polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 6,171,820 and 6,238,884), the skilled artisan can produce DNAsequences that encode TWEAKR polypeptides comprising various additionsor substitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences, including TWEAKR fragments,variants, derivatives, and fusion polypeptides.

Transgenic animals, including mice, goats, sheep, and pigs, andtransgenic plants, including tobacco, tomato, legumes, grasses, andgrains, may also be used as bioreactors for the production of TWEAKRpolypeptides, including soluble TWEAKR polypeptides. In the case oftransgenic animals, it is particularly advantageous to construct achimeric DNA including a TWEAKR coding sequence operably linked tocis-acting regulatory sequences that promote expression of the solubleTWEAKR in milk and/or other body fluids (see, e.g., U.S. Pat. No.5,843,705; U.S. Pat. No. 5,880,327). In the case of transgenic plants itis particularly advantageous to produce TWEAKR in a particular celltype, tissue, or organ (see, e.g., U.S. Pat. No. 5,639,947; U.S. Pat.No. 5,889,189).

The skilled artisan will recognize that the procedure for purifyingexpressed soluble TWEAKR polypeptides will vary according to the hostsystem employed, and whether or not the recombinant polypeptide issecreted. Soluble TWEAKR polypeptides may be purified using methodsknown in the art, including one or more concentration, salting-out, ionexchange, hydrophobic interaction, affinity purification, HPLC, or sizeexclusion chromatography steps. Fusion polypeptides comprising Fcmoieties (and multimers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Methods of Treatment

Described below are methods and compositions employing the TWEAKreceptor or ligand, or the genes encoding the TWEAK receptor or ligand,to promote or suppress angiogenesis in a subject, a target tissue, or agroup of cells. The terms “treat,” “treating,” “treatment,” “therapy,”“therapeutic,” and the like are intended to include preventativetherapy, prophylactic therapy, ameliorative therapy, and curativetherapy. By “subject” is meant any mammal (e.g., bovine, equine,porcine, canine, feline, and primates), but preferably is a human.

The disclosed polypeptides, compositions, and methods are used toinhibit angiogenesis, modulate cell migration and/or proliferation, orother TWEAKR-mediated responses in a subject in need of such treatment.The term “TWEAKR-mediated response” includes any cellular,physiological, or other biological response that is caused at least inpart by the binding of TWEAK ligand to TWEAKR, or which may be inhibitedor suppressed, in whole or in part, by blocking TWEAK from binding toTWEAKR. The treatment is advantageously administered in order to preventthe onset or the recurrence of a disease or condition mediated byangiogenesis, or to treat a subject that has a disease or conditionmediated by angiogenesis. Diseases and conditions mediated byangiogenesis include but are not limited to ocular disorders, malignantand metastatic conditions, and inflammatory diseases. In some instancesstimulation of a TWEAK-TWEAKR response may be beneficial (e.g., duringtissue or would repair). Accordingly, administration of TWEAK or TWEAKRin such tissue or cells may be used to promote wound repair.

Among the ocular disorders that can be treated according to the presentinvention are eye diseases characterized by ocular neovascularizationincluding, but not limited to, diabetic retinopathy (a majorcomplication of diabetes), retinopathy of prematurity (this devastatingeye condition, that frequently leads to chronic vision problems andcarries a high risk of blindness, is a severe complication during thecare of premature infants), neovascular glaucoma, retinoblastoma,retrolental fibroplasia, rubeosis, uveitis, macular degeneration, andcorneal graft neovascularization. Other eye inflammatory diseases,ocular tumors, and diseases associated with choroidal or irisneovascularization can also be treated according to the presentinvention.

The present invention can also be used to treat cell proliferativedisorders, including malignant and metastatic conditions such as solidtumors. Solid tumors include both primary and metastatic sarcomas andcarcinomas.

The present invention can also be used to treat inflammatory diseasesincluding, but not limited to, arthritis, rheumatism, and psoriasis.

Other diseases and conditions that can be treated according to thepresent invention include benign tumors and preneoplastic conditions,myocardial angiogenesis, hemophilic joints, scleroderma, vascularadhesions, atherosclerotic plaque neovascularization, telangiectasia,and wound granulation.

Disease states that are angiogenic-dependent include coronary orperipheral atherosclerosis and ischemia of any tissue or organ,including the heart, liver, brain, and the like. These types of diseasescan be treated by compositions that promote angiogenesis.

In addition to polypeptides comprising a fragment of TWEAKRextracellular domain, soluble TWEAKR multimers, and antibodies that bindto the TWEAKR extracellular domain, other forms of TWEAKR antagonistscan also be administered to achieve a therapeutic effect. Examples ofother forms of TWEAKR antagonists include other antibodies such asantibodies against TWEAK, antisense nucleic acids, ribozymes, muteins,aptamers, and small molecules directed against TWEAKR or against TWEAK.

The methods according to the present invention can be tested in in vivoanimal models to confirm the desired prophylactic or therapeuticactivity, as well as to determine the optimal therapeutic dosage, priorto administration to humans.

The amount of a particular TWEAKR antagonist that will be effective in aparticular method of treatment depends upon age, type and severity ofthe condition to be treated, body weight, desired duration of treatment,method of administration, and other parameters. Effective dosages aredetermined by a physician or other qualified medical professional.Typical effective dosages are about 0.01 mg/kg to about 100 mg/kg bodyweight. In some preferred embodiments the dosage is about 0.1-50 mg/kg;in some preferred embodiments the dosage is about 0.5-10 mg/kg. Thedosage for local administration is typically lower than for systemicadministration. In some embodiments a single administration issufficient; in some embodiments the TWEAKR antagonist is administered asmultiple doses over one or more days.

The TWEAKR antagonists are typically administered in the form of apharmaceutical composition comprising one or more pharmacologicallyacceptable carriers. Pharmaceutically acceptable carriers includediluents, fillers, adjuvants, excipients, and vehicles that arepharmaceutically acceptable for the route of administration, and may beaqueous or oleaginous suspensions formulated using suitable dispersing,wetting, and suspending agents.

Pharmaceutically acceptable carriers are generally sterile and free ofpyrogenic agents, and may include water, oils, solvents, salts, sugarsand other carbohydrates, emulsifying agents, buffering agents,antimicrobial agents, and chelating agents. The particularpharmaceutically acceptable carrier and the ratio of active compound tocarrier are determined by the solubility and chemical properties of thecomposition, the mode of administration, and standard pharmaceuticalpractice.

The compositions as described herein may be contained in a vial, bottle,tube, syringe inhaler or other container for single or multipleadministrations. Such containers may be made of glass or a polymermaterial such as polypropylene, polyethylene, or polyvinylchloride, forexample. Preferred containers may include a seal or other closuresystem, such as a rubber stopper that may be penetrated by a needle inorder to withdraw a single dose and then re-seal upon removal of theneedle. All such containers for injectable liquids, lyophilizedformulations, reconstituted lyophilized formulations or reconstitutablepowders for injection known in the art or for the administration ofaerosolized compositions are contemplated for use in the presentlydisclosed compositions and methods.

The TWEAKR antagonists are administered to the subject in a mannerappropriate to the indication. Thus, for example, a TWEAKR antagonist,or a pharmaceutical composition thereof, may be administered byintravenous, transdermal, intradermal, intraperitoneal, intramuscular,intranasal, epidural, oral, topical, subcutaneous, intracavity,sustained release from implants, peristaltic routes, or by any othersuitable technique. Parenteral administration is preferred.

In certain embodiments of the claimed invention, the treatment furthercomprises treating a subject with one or more additional agents such asadditional chemotherapeutic agents. The additional chemotherapeuticagent(s) may be administered prior to, concurrently with, or followingthe administration of the TWEAKR antagonist. The use of more than onechemotherapeutic agent is particularly advantageous when the subjectthat is being treated has a solid tumor. In some embodiments of theclaimed invention, the treatment further comprises treating the subjectwith radiation. Radiation, including brachytherapy and teletherapy, maybe administered prior to, concurrently with, or following theadministration of the second chemotherapeutic agent(s) and/or TWEAKRantagonist.

When the subject that is being treated has a solid tumor, the methodpreferably includes the administration of, in addition to a TWEAKRantagonist, one or more chemotherapeutic agents selected from the groupconsisting of alkylating agents, antimetabolites, vinca alkaloids andother plant-derived chemotherapeutics, nitrosoureas, antitumorantibiotics, antitumor enzymes, topoisomerase inhibitors, platinumanalogs, adrenocortical suppressants, hormones, hormone agonists andantagonists, antibodies, immunotherapeutics, blood cell factors,radiotherapeutics, and biological response modifiers.

In some preferred embodiments the method includes administration of, inaddition to a TWEAKR antagonist, one or more chemotherapeutic agentsselected from the group consisting of cisplatin, cyclophosphamide,mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil,5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine,and vinblastine, lymphokines and cytokines such as interleukins,interferons (including alpha, beta, or delta), and TNF, chlorambucil,busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine,cytarabine, mercaptopurine, thioguanine, vindesine, etoposide,teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine,mitotane, tamoxifen, and fluoxymesterone.

In some preferred embodiments the method includes administration of, inaddition to a TWEAKR antagonist, one or more chemotherapeutic agents,including various soluble forms thereof, selected from the groupconsisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12,4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptorantagonists, TRAIL, VEGF antagonists, VEGF receptor (including VEGF-R1and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, Tekantagonists, and CD148 (also referred to as DEP-1, ECRTP, and PTPRJ, seeTakahashi et al., J. Am. Soc. Nephrol. 10:2135-45, 1999) agonists. Insome preferred embodiments the TWEAKR antagonists of the invention areused as a component of, or in combination with, “metronomic therapy,”such as that described by Browder et al. and Klement et al. (CancerResearch 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see alsoBarinaga, Science 288:245, 2000).

The polypeptides, compositions, and methods of the present invention maybe used as a first line treatment, for the treatment of residual diseasefollowing primary therapy, or as an adjunct to other therapies includingchemotherapy, surgery, radiation, and other therapeutic methods known inthe art.

When the nucleic acid sequences of the present invention are deliveredaccording to the methods disclosed herein, it is advantageous to use adelivery mechanism so that the sequences will be incorporated into acell for expression. Delivery systems that may advantageously beemployed in the contemplated methods include the use of, for example,viral delivery systems such as retroviral and adenoviral vectors, aswell as non-viral delivery systems. Such delivery systems are well knownby those skilled in the art.

Methods of Screening

TWEAKR as described herein may be used in a variety of methods ofscreening to isolate, for example, TWEAKR agonists and antagonists.TWEAKR agonists are compounds that promote the biological activity ofTWEAKR and TWEAKR antagonists are compounds that inhibit the biologicalactivity of TWEAKR. Compounds identified via the following screeningassays can be used in compositions and methods for modulatingangiogenesis to treat a variety of disease states. The present inventionprovides methods of screening for compounds that (1) modulate TWEAKR orligand gene expression in a target tissue or cell, (2) modulate theTWEAKR-ligand interaction to regulate angiogenesis; (3) bind to theTWEAKR or ligand to influence angiogenesis; or (4) interfere with orregulate the bound TWEAKR-ligand complex's influence on downstreamevents such as angiogenesis. Accordingly, the polypeptides, andfragments thereof, of the invention can be used to regulate, influence,and modulate (i.e., increase or decrease) a biological activityassociated with interaction of TWEAK or TWEAKR with its cognate.

The present invention contemplates the use of assays that are designedto identify compounds that modulate the activity of a TWEAKR or ligandgene (e.g., modulate the level of TWEAKR or TWEAK gene expression and/ormodulate the level of TWEAKR or TWEAK gene product activity). Assays mayadditionally be utilized that identify compounds that bind to TWEAKR orTWEAK gene regulatory sequences (e.g., promoter sequences; see e.g.Platt, 1994, J. Biol. Chem. 269, 28558-28562), and that may modulate thelevel of TWEAKR or TWEAK gene expression.

Such an assay may involve, for example, the use of a control system, inwhich transcription and translation of the TWEAKR or ligand gene occurs,in comparison to a system including a test agent suspected ofinfluencing normal transcription or translation of a TWEAKR or ligandgene. For example, one could determine the rate of TWEAKR RNA producedby cardiac cells, and use this to determine if a test agent influencesthat rate. To assess the influence of a test agent suspected toinfluence this normal rate of transcription, one would first determinethe rate of TWEAKR RNA production in a cardiac cell culture by, forexample, Northern Blotting. One could then administer the test agent toa cardiac cell culture under otherwise identical conditions as thecontrol culture. The rate of TWEAKR RNA in the culture treated with thetest agent could be determined by, for example, Northern Blotting, andcompared to the rate of TWEAKR RNA produced by the control culturecells. An increase in the TWEAKR RNA in the cells contacted with thetest agent relative to control cells is indicative of a stimulator ofTWEAKR gene transcription in cardiac cells, while a decrease isindicative of an inhibitor of TWEAKR gene transcription in cardiaccells.

There are a variety of other methods that can be used to determine thelevel of TWEAKR or ligand gene expression as well, and may further beused in assays to determine the influence of a test agent on the levelof TWEAKR or ligand gene expression. For example, RNA from a cell typeor tissue known, or suspected, to express the TWEAK receptor or ligandgene, such as cardiac tissue, may be isolated and tested utilizinghybridization or PCR techniques. The isolated cells can be derived fromcell culture or from a subject. The analysis of cells taken from culturemay be a necessary step in the assessment of cells to be used as part ofa cell-based gene therapy technique or, alternatively, to test theeffect of compounds on the expression of the TWEAK receptor or ligandgene. Such analyses may reveal both quantitative and qualitative aspectsof the expression pattern of the TWEAK receptor or ligand gene,including activation or inactivation of TWEAKR or ligand geneexpression.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). A sequence within the cDNAis then used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the TWEAKR or ligand gene nucleic acidsegments described above. The preferred lengths of such nucleic acidreagents are at least 9-30 nucleotides. For detection of the amplifiedproduct, the nucleic acid amplification may be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product may be made such that the product may bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

Additionally, it is possible to perform such TWEAKR or ligand geneexpression assays “in situ”, i.e., directly upon tissue sections (fixedand/or frozen) of subject tissue obtained from biopsies or resections,such that no nucleic acid purification is necessary. TWEAKR or ligandgene nucleic acid segments described above can be used as probes and/orprimers for such in situ procedures (see, for example, Nuovo, G. J.,1992, “PCR In situ Hybridization: Protocols And Applications”, RavenPress, NY).

Compounds identified via assays such as those described herein may beuseful, for example, in modulating angiogenesis influenced by TWEAKR orTWEAKR-ligand interaction. Such methods of stimulating or inhibitingTWEAK- or TWEAKR-influenced angiogenesis are discussed herein.

Alternatively, assay systems may be designed to identify compoundscapable of binding the TWEAKR or ligand polypeptide of the invention andthereby influencing angiogenesis resulting from this interaction.Compounds identified may be useful, for example, in modulating thevascularization of target tissues or cells, may be utilized in screensfor identifying compounds that disrupt normal TWEAKR-ligandinteractions, or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to theTWEAK receptor or ligand involves preparing a reaction mixture of theTWEAK receptor or ligand and the test agent under conditions and for atime sufficient to allow the two components to interact or bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay screening for compoundsthat bind to the TWEAK receptor, would involve anchoring the TWEAKreceptor or the test substance onto a solid phase and detectingTWEAKR/test agent complexes anchored on the solid phase at the end ofthe reaction. In one embodiment of such a method, the TWEAK receptor maybe anchored onto a solid surface, and the test agent, which is notanchored, may be labeled, either directly or indirectly. Alternatively,these same methods could be used to screen for test agents that bind tothe TWEAK ligand rather than receptor.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g. by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously non-immobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g. using an immobilized antibody specific for the TWEAKreceptor or ligand or the test agent to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

Those compounds identified as binding agents for either the TWEAKreceptor or the TWEAK ligand may further be assessed for their abilityto interfere with TWEAKR-ligand interaction, as described below, andthereby suppress or promote angiogenesis resulting from TWEAKR-ligandinteraction. Such compounds may then be used therapeutically tostimulate or inhibit angiogenesis.

The TWEAKR and ligand polypeptides of the present invention may also beused in a screening assay to identify compounds and small moleculeswhich specifically interact with the disclosed TWEAK receptor or ligandto either inhibit (antagonize) or enhance (agonize) interaction betweenthese molecules. Thus, for example, polypeptides of the invention may beused to identify antagonists and agonists from cells, cell-freepreparations, chemical libraries, and natural product mixtures. Theantagonists and agonists may be natural or modified substrates, ligands,enzymes, receptors, and the like, of the polypeptides of the instantinvention, or may be structural or functional mimetics of thepolypeptides. Potential antagonists of the TWEAKR-ligand interaction ofthe instant invention may include small molecules, polypeptides,peptides, peptidomimetics, and antibodies that bind to and occupy abinding site of the polypeptides, causing them to be unavailable tointeract and therefore preventing their normal ability to modulateangiogenesis. Other potential antagonists are antisense molecules thatmay hybridize to mRNA in vivo and block translation of the mRNA into thepolypeptides of the instant invention. Potential agonists include smallmolecules, polypeptides, peptides, peptidomimetics, and antibodies thatbind to the instant TWEAKR and TWEAK polypeptides and influenceangiogenesis as caused by the disclosed interactions of the TWEAKR andTWEAK polypeptides of the instant invention.

Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physiochemical andpharmacological properties that enhance cell penetration, resistdegradation and prolong their physiological half-lives. (Gibbs,“Pharmaceutical Research in Molecular Oncology,” Cell, Vol. 79, (1994)).

Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, may be used to bind to and inhibit thepolypeptides of the instant invention by blocking the commencement of asignaling cascade. It is preferable that the antibodies are humanized,and more preferable that the antibodies are human. The antibodies of thepresent invention may be prepared by any of a variety of well-knownmethods. Alternatively, antibodies may bind to and activate thepolypeptides of the instant by mimicking the interaction of apolypeptide of the invention with its cognate. One of skill in the artusing the assay methods and techniques herein can determine whether anantibody is an antagonist or agonist.

Specific screening methods are known in the art and many are extensivelyincorporated in high throughput test systems so that large numbers oftest agents can be screened within a short amount of time. The assayscan be performed in a variety of formats, including protein-proteinbinding assays, biochemical screening assays, immunoassays, cell basedassays, and the like. These assay formats are well known in the art. Thescreening assays of the present invention are amenable to screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides and other antagonists andagonists.

One embodiment of a method for identifying molecules which antagonize orinhibit TWEAKR-ligand interaction involves adding a candidate moleculeto a medium which contains cells that express the polypeptides of theinstant invention; changing the conditions of said medium so that, butfor the presence of the candidate molecule, the polypeptides wouldinteract; and observing the binding and inhibition of angiogenesis.Binding of the TWEAK receptor and ligand can be determined according tocompetitive binding assays outlined above, and well known in the art.The angiogenic effect of this binding can be determined via cellproliferation assays such as, for example, cell density assays, cornealpocket assays, or other cell proliferation assays that are alsowell-known in the art. The activity of the cells contacted with thecandidate molecule may then be compared with the identical cells, whichwere not contacted, and agonists and antagonists of the TWEAKpolypeptide interactions of the instant invention may be identified. Themeasurement of biological activity may be performed by a number ofwell-known methods such as measuring the amount of protein present (e.g.an Enzyme-Linked Immunosorbent Assay (ELISA)), production of cytokines(e.g., IL-8 and IL-6; see, e.g., Saas et al., Glia 32(1):102-7, 2000),or of the protein's activity. A decrease in biological stimulation oractivation would indicate an antagonist. An increase would indicate anagonist.

Screening assays can further be designed to find molecules that mimicthe biological activity resulting from the TWEAKR and/or TWEAKpolypeptide interactions of the instant invention. Molecules which mimicthe biological activity of a polypeptide may be useful for enhancing thebiological activity of the polypeptide. To identify compounds fortherapeutically active agents that mimic the biological activity of apolypeptide, it must first be determined whether a candidate moleculebinds to the polypeptide. A binding candidate molecule is then added toa biological assay to determine its biological effects. The biologicaleffects of the candidate molecule are then compared to those of thepolypeptide.

Additionally, complex formation within reaction mixtures containing thetest agent and normal TWEAKR or ligand gene protein may also be comparedto complex formation within reaction mixtures containing the test agentand a mutant TWEAKR or ligand gene protein. This comparison may beimportant in those cases wherein it is desirable to identify compoundsthat disrupt interactions of mutant but not normal TWEAKR or ligand geneproteins.

The assay for compounds that interfere with the interaction of theTWEAKR or ligand gene products and binding partners can be conducted ina heterogeneous or homogeneous format. Heterogeneous assays involveanchoring either TWEAKR or ligand gene product or the binding partneronto a solid phase and detecting complexes anchored on the solid phaseat the end of the reaction. In homogeneous assays, the entire reactionis carried out in a liquid phase. In either approach, the order ofaddition of reactants can be varied to obtain different informationabout the compounds being tested. For example, test agents thatinterfere with the interaction between the TWEAKR or ligand geneproducts and the binding partners, e.g. by competition, can beidentified by conducting the reaction in the presence of the testsubstance; e.g. by adding the test substance to the reaction mixtureprior to or simultaneously with the TWEAKR and ligand gene products.Alternatively, test agents that disrupt preformed complexes, e.g.compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test agent tothe reaction mixture after complexes have been formed.

In a particular embodiment, the TWEAKR or ligand gene product can beprepared for immobilization using recombinant DNA techniques. Forexample, the TWEAKR or ligand coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive binding partner can bepurified and used to raise a monoclonal antibody, using methodsroutinely practiced in the art. This antibody can be labeled with theradioactive isotope ¹²⁵I, for example, by methods routinely practiced inthe art. In a heterogeneous assay, e.g. the GST-TWEAKR or ligand fusionprotein can be anchored to glutathione-agarose beads. The TWEAKR orligand gene product can then be added in the presence or absence of thetest agent in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between the TWEAKRand ligand gene products can be detected by measuring the amount ofradioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test agent willresult in a decrease in measured radioactivity.

Alternatively, a GST-TWEAKR gene fusion protein and TWEAK ligand geneproduct (or vice versa) can be mixed together in liquid in the absenceof the solid glutathione-agarose beads. The test agent can be addedeither during or after the species is allowed to interact. This mixturecan then be added to the glutathione-agarose beads and unbound materialis washed away. Again the extent of inhibition of the TWEAKR-ligand geneproduct interaction can be detected by adding the labeled antibody andmeasuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the TWEAKR and/or ligand protein, in place of one or both of thefull-length proteins. Any number of methods routinely practiced in theart can be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the segmentscan be engineered to express peptide fragments of the protein, which canthen be tested for binding activity and purified or synthesized.

As an example, and not by way of limitation, a TWEAKR or ligand geneproduct can be anchored to a solid material, as described above, bymaking a GST-TWEAKR or ligand fusion protein and allowing it to bind toglutathione agarose beads. The interactive binding partner obtained canbe labeled with a radioactive isotope, such as ³⁵S, and cleaved with aproteolytic enzyme such as trypsin. Cleavage products can then be addedto the anchored GST-TWEAKR fusion protein or TWEAK ligand fusion proteinand allowed to bind. After washing away unbound peptides, labeled boundmaterial, representing the binding partner binding domain, can beeluted, purified, and analyzed for amino acid sequence by well-knownmethods. Peptides so identified can be produced synthetically or fusedto appropriate facilitative proteins using recombinant DNA technology.

The TWEAKR-ligand interactions of the invention, in vivo, initiate acascade of events that either stimulate or suppress angiogenesis in atarget group of cells or tissues. Molecules, such as nucleic acidmolecules, proteins, or small molecules may, in turn, influence thiscascade. Compounds that disrupt the TWEAKR-ligand interaction may beuseful in regulating angiogenesis.

The basic principle of the assay systems used to identify compounds thatinterfere with the angiogenic or anti-angiogenic effect of TWEAKR-ligandinteraction involves preparing a reaction mixture containing the TWEAKreceptor and ligand under conditions and for a time sufficient to allowthe two to interact or bind, thus forming a complex. In order to test acompound for inhibitory activity of the effect of this interaction, thereaction mixture is prepared in the presence and absence of the testagent. The test agent may be initially included in the reaction mixture,or may be added at a time subsequent to the addition of theTWEAKR-ligand complex. Control reaction mixtures are incubated withoutthe test agent or with a placebo. The inhibition or potentiation of anyeffect of the TWEAK complex on vascularization is then detected. Normalangiogenic response in the control reaction, but not in the reactionmixture containing the test agent, indicates that the compoundinterferes with the cascade of events initiated by the TWEAKR-ligandinteraction. Enhanced angiogenesis in the test agents-containing cultureindicates a stimulator of the TWEAKR-ligand complex effect.

In another embodiment, the techniques of rational drug design can beused to develop TWEAKR binding agents (e.g., agonist or antagonists ofTWEAKR). The goal of rational drug design is to produce structuralanalogs of biologically active polypeptides of interest or of smallmolecules with which they interact, e.g., substrates, binding agents,inhibitors, agonists, antagonists, and the like. The methods providedherein can be used to fashion or identify agents which are more activeor stable forms of the polypeptide or which enhance or interfere withthe function of a polypeptide in vivo (Hodgson J (1991) Biotechnology9:19-21, incorporated herein by reference). In one approach, thethree-dimensional structure of a TWEAKR polypeptide of the invention, aligand or binding partner, or of a polypeptide-binding partner complex,is determined by x-ray crystallography, by nuclear magnetic resonance,or by computer homology modeling or, most typically, by a combination ofthese approaches. Both the shape and charges of the polypeptide areascertained to elucidate the structure and to determine active site(s)or sites of interaction of the molecule. Relevant structural informationis used to design analogous molecules, to identify efficient inhibitors,or to identify small molecules that may bind to a polypeptide of theinvention. Useful examples of rational drug design may include moleculeswhich have improved activity or stability as shown by Braxton S andWells J A (1992 Biochemistry 31:7796-7801) or which act as inhibitors,agonists, or antagonists of native peptides as shown by Athauda S B etal. (1993 J Biochem 113:742-746), incorporated herein by reference. Theuse of TWEAKR or TWEAK polypeptide structural information in molecularmodeling software systems provides for the design of inhibitors orbinding agents useful in modulating TWEAKR-TWEAK interactions orbiological activity. A particular method of the invention comprisesanalyzing the three dimensional structure of TWEAK or TWEAKRpolypeptides for likely binding/interaction sites of substrates orligands, synthesizing a new molecule that incorporates a predictivereactive site, and assaying the new molecule as described furtherherein. Examples of algorithms, software, and methods for modelingsubstrates or binding agents based upon the three-dimensional structureof a protein are described in PCT publication WO107579A2, entitled“METHODS AND COMPOSITIONS FOR DETERMINING ENZYMATIC ACTIVITY,” thedisclosure of which is incorporated herein.

EXAMPLES

The following examples are intended to illustrate particular embodimentsand not to limit the scope of the invention.

Example 1 Identification of the TWEAK Receptor

Expression Cloning of TWEAKR cDNA

To clone TWEAKR cDNA, an expression vector encoding a growth hormoneleader, a leucine zipper multimerization domain, and the C-terminalextracellular domain of human TWEAK (see Chicheportiche et al., J. Biol.Chem. 272(51):32401, 1997) was constructed. This expression vector,which was named pDC409-LZ-TWEAK, comprised the DNA sequence SEQ ID NO: 1and encoded the polypeptide SEQ ID NO:2. pDC409-LZ-TWEAK conditionedsupernatants were produced by transient transfection into CV1-EBNAcells. These supernatants were incubated with magnetic beads coated withpolyclonal goat anti-mouse antibody that had previously been incubatedwith a mouse monoclonal antibody against the leucine zipper. Controlbeads were produced by mixing the coated beads with supernatants fromcells transfected with empty vector.

A monolayer of COS cells grown in a T175 flask was transfected with 15μg of DNA pools of complexity of 100,000 from a human umbilical veinendothelial cell (HUVEC) cDNA expression library. After 2 days thesecells were lifted from the flask, and incubated in 1.5 mls of bindingmedia plus 5% non-fat dried milk for 3 hours at 4° C. on a rotatorwheel. Cells were pre-cleared by adding control beads and rotated at 4degrees ° C. for an additional 45 minutes after which bead bound cellswere removed with a magnet. Pre-clearing was repeated 2-3 times, thenTWEAK coated beads were added to the cells and rotated 30 minutes at 4°C. Cells binding the TWEAK beads were separated by use of a magnet andwashed 4× in phosphate buffered saline (PBS). Plasmid DNA was extractedfrom these cells by lysing in 0.1% SDS, and electroporating thesupernatants in DH101B cells. Colonies were grown overnight onampicillin selective media. Transformants were pooled and used as asource of plasmid DNA for a further round of panning. After 2 rounds ofpanning, positive clones were picked from the resulting pool based ontheir ability to bind TWEAK using a slide binding protocol. Slidebinding was performed as described more fully below, with the exceptionthat TWEAKR positive slides were detected by incubation with TWEAKRconditioned supernatants followed by incubation with ¹²⁵I-labeled M15anti-leucine zipper.

The human TWEAK receptor (also called TWEAKR) cDNA was determined tohave the sequence SEQ ID NO:3, which encodes a 129 residue polypeptide(SEQ ID NO:4). Examination of the sequence predicts a polypeptide havingan approximately 80 amino acid extracellular domain (residues 1-80 ofSEQ ID NO:4, including the signal peptide, amino acids 1-27), anapproximately 20 amino acid transmembrane domain (residues 81-100 of SEQID NO:4), and an approximately 29 amino acid intracellular domain(residues 101-129 of SEQ ID NO:4). TWEAKR is the smallest known TNFreceptor family member. It has a single cysteine-rich repeat region inthe extracellular domain, as compared to the 3-4 repeats of other TNFreceptor family members. The TWEAKR polypeptide was previously describedas a transmembrane protein encoded by a human liver cDNA clone (WO98/55508, see also WO 99/61471), but had not been identified as theTWEAK receptor. A murine homolog, the FGF-inducible Fn14 (Meighan-Manthaet al., J. Biol. Chem. 274(46):33166, 1999), is approximately 82%identical to the human protein, as shown by the alignment in FIG. 1.

The newly identified TWEAK receptor was tested side by side with DR3(which had been identified as the TWEAK receptor by Marsters et al.,Current Biology 8:525, 1998) for the ability to bind to TWEAK.

TWEAKR Binds to TWEAK

Slides of COS cells were transfected with expression vectors containingTWEAKR, DR3, or vector without insert (control). After two days thecells were incubated with concentrated supernatants from CV-1 cellstransfected with a vector encoding the leucine zipper TWEAKextracellular domain fusion protein. One hour later the cells werewashed and probed with an ¹²⁵I labeled antibody against theleucine-zipper domain. The slides were washed, fixed, andautoradiography was performed using x-ray film. The TWEAKR transfectedcells bound significant amounts of TWEAK. TWEAK did not bind to thecells transfected with DR3 or the control cells. This experimentconfirmed that the TWEAKR polypeptide identified in part A above, ratherthan DR3, is the major receptor for TWEAK. After discovery of thefunctional TWEAK receptor, other investigators also reported that DR3 isnot the major receptor for TWEAK (Kaptein et al., FEBS Lett.,485(2-3):135, 2000. The TWEAK-TWEAKR binding interaction was furthercharacterized by Scatchard analysis.

CV-1 cells were transfected with human full length TWEAK and mixed 1:30with Raji cells, which do not express TWEAK. The cells were incubatedwith serial dilutions of 125-I labeled human TWEAKR-Fc for 2 hours at 4degrees Celsius. Free and bound probe was separated by microfuging thesamples through a phalate oil mixture in plastic tubes. Supernatants andpellets were gamma-counted. Scatchard analyses of TWEAK ligand bindingthe TWEAK receptor showed a binding affinity constant (Ka) ofapproximately 4.5×10⁸ M⁻¹.

The TWEAK Receptor is Strongly Expressed in Cardiac Tissue

To determine the expression pattern of the TWEAK receptor, Northern blotanalyses were performed. Human multiple tissue northern blots werepurchased from Clontech (Palo Alto, Calif.) and probed with ³²P labeledrandom primed DNA from the TWEAKR coding region. The blots were washedand autoradiography was performed using x-ray film. Results showed thatin the adult TWEAKR is strongly expressed in heart, placenta, and someskeletal muscle samples. Strong expression in heart tissue furthersupports the utility of TWEAKR in the diagnosis and treatment of cardiacdisease. In contrast to the adult, the fetal tissues expressed TWEAKRmore ubiquitously; TWEAKR transcripts were seen in the lung and liver.

Example 2 Preparation of TWEAKR Antagonists and Agonists

Because TWEAK induces angiogenesis, TWEAKR agonists (such as agonisticantibodies) may be used to promote angiogenesis and TWEAKR antagonists(such as soluble receptors and antagonistic antibodies) may be used toinhibit angiogenesis.

Recombinant Production of Soluble TWEAKR-Fc Fusion Polypeptides

To construct a nucleic acid encoding the TWEAKR extracellular domainfused to Fc, a nucleic acid encoding the N-terminal 79 amino acids fromTWEAKR, including the leader (signal peptide), was joined to a nucleicacid encoding an Fc portion from human IgG1. Sequences for thisconstruct are shown as SEQ ID NO:6 and 8 (nucleic acid) and SEQ ID NO:7and 9 (amino acid). In SEQ ID NO:7 and 9, residues 1-27 are thepredicted signal peptide (predicted to be cleaved upon secretion fromthe cell; the actual cleavage site was identified by N-terminal sequenceanalysis, see below), residues 28-79 and 28-70 of SEQ ID NO:7 and 9,respectively, are from the cysteine-rich TWEAKR extracellular domain,residues 80-81 and 71-72 of SEQ ID NO:7 and 9, respectively, are from aBglII cloning site, and the remainder is the Fc portion. Upon insertioninto a mammalian expression vector, and expression in and secretion froma mammalian host cells, these construct produced a polypeptidedesignated TWEAKR-Fc (SEQ ID NO:7) and TWEAKR-FcΔ9 (SEQ ID NO:9).N-terminal sequence analysis determined that the secreted polypeptidesdesignated TWEAKR-Fc and TWEAKR-FcΔ9 had an N-terminus corresponding toresidue 28 (Glu) of SEQ ID NO:7 and 9, respectively. Anti-angiogenicactivity of TWEAKR-Fc was demonstrated using assays such as thosedescribed in the following examples. An analogous Fc-fusion constructwas prepared using the murine TWEAKR extracellular domain.

The extracellular domain of human TWEAKR was expressed in E. coli as aleucine zipper dimer fusion protein. A cDNA was constructed with the aidof PCR to place an initiator Met residue next to TWEAKR DNA encodingamino acids Glu28 to Trp79. In addition, cDNA sequences were added thatencoded Flag and the leucine zipper dimer at the C-terminal end. ThecDNA was then ligated into an E. coli expression vector. The vector wasdesigned to express recombinant protein upon induction in E. coli.Several promoters or transcriptional control units can be used includingthe T₇ promoter, the P_(L) promoter, and the Tac promoter. A number ofcommercially available vectors are known in the art.

E. coli cells containing the TWEAKR-Flag-LeuZip2 were cultured andinduced for expression. After several hours, E. coli cells werecollected and lysed to release intracellular proteins. The E. colilysate was fractionated on SDS-PAGE and Western blotted for the Flagantigen. A specific Flag reactive band was seen at approximately 12.5kDa, the expected size of the TWEAKR-Flag-LeuZip2. Additional blots wereprobed with TWEAK and bands visualized with an anti-TWEAK antibody. Thesame 12.5 kDa band was visualized indicating the E. coli-expressedTWEAKR is able to bind its ligand.

Production of Antibodies that Bind the TWEAKR Extracellular Domain

BALB/c mice are immunized with TWEAKR extracellular domain and spleencells are collected and used to prepare hybridomas using standardprocedures. Hybridoma supernatants are screened, using ELISA, for theability to bind TWEAKR. Positives are cloned two times, to insuremonoclonality, then isotyped and reassayed for reactivity to TWEAKR.Antibodies and antibody derivatives are also prepared using transgenicmice that express human immunoglobulins and through the use of phagedisplay. The resulting antibodies are tested in assays such as thosedescribed in the examples below, to characterize their ability tomodulate the TWEAK-TWEAKR interaction, TWEAKR signaling, angiogenesis,and other downstream biological activities.

Agonistic antibodies are used to promote TWEAK-induced biologicalactivities such as angiogenesis, and antagonistic antibodies are used toinhibit TWEAK-induced biological activities such as angiogenesis. Forsome applications, the activity of antagonistic antibodies is augmentedby conjugation to a radioisotope, to a plant-, fungus-, orbacterial-derived cytotoxin such as ricin A or diphtheria toxin, or toanother chemical poison. And because of the restricted tissuedistribution of TWEAKR, antibodies that bind to TWEAKR are particularlyuseful as targeting agents for imaging or delivering therapeutics to thevasculature. Antibodies that bind TWEAKR can be used, for example, totarget a detectable label or chemotherapeutic to the mural cells(pericytes and vascular smooth muscle cells). Detectable labels mayinclude radioisotopes, chemiluminescent and fluorescent compounds, andenzymes. These techniques are useful, for example, in the diagnosis,staging, and treatment of neoplasms.

Example 3 Activity of TWEAKR-Fc in a Wound Closure Assay

A planar endothelial cell migration (wound closure) assay was used toquantitate the inhibition of angiogenesis by TWEAKR-Fc in vitro. In thisassay, endothelial cell migration is measured as the rate of closure ofa circular wound in a cultured cell monolayer. The rate of wound closureis linear, and is dynamically regulated by agents that stimulate andinhibit angiogenesis in vivo.

Primary human renal microvascular endothelial cells (HRMEC) wereisolated, cultured, and used at the third passage after thawing, asdescribed in Martin et al., In vitro Cell Dev Biol 33:261, 1997.Replicate circular lesions, “wounds,” (600-800 micron diameter) weregenerated in confluent HRMEC monolayers using a silicon-tipped drillpress. At the time of wounding the medium (Dulbecco's Modified eagleMedium (DMEM)+1% bovine serum albumin (BSA)) was supplemented with 20ng/ml PMA (phorbol-12-myristate-13-acetate), EGF (4 ng/ml), and 0.150 to5 μg/ml TWEAKR-Fc, or a combination of 40 ng/ml EGF and 0.150 to 5 μg/mlTWEAKR-Fc. As a control for TWEAKR-Fc indicated samples received 5 μg/mlIgG-Fc. The residual wound area was measured as a function of time (0-12hours) using a microscope and image analysis software (Bioquant,Nashville, Tenn.). The relative migration rate was calculated for eachagent and combination of agents by linear regression of residual woundarea plotted over time. The results are shown in FIGS. 2-3.

Compared to huIgG or media+BSA, TWEAKR-Fc inhibited PMA-inducedendothelial migration in a dose responsive manner, reducing the rate ofmigration to unstimulated levels at 1.5 to 5 μg/ml (FIG. 2). NeitherhuIgG nor TWEAKR-Fc inhibited basal (uninduced) migration. When HRMECmigration was induced by EGF, TWEAKR-Fc inhibited endothelial migrationin a dose-dependent manner, reducing the rate of migration tounstimulated levels at 5 μg/ml (FIG. 3).

Example 4 Activity of TWEAKR-Fc In a Corneal Pocket Assay

A mouse corneal pocket assay was used to quantitate the inhibition ofangiogenesis by TWEAKR-Fc in vivo. In this assay, agents to be testedfor angiogenic or anti-angiogenic activity are immobilized in a slowrelease form in a hydron pellet, which is implanted into micropocketscreated in the corneal epithelium of anesthetized mice. Vascularizationis measured as the appearance, density, and extent of vessel ingrowthfrom the vascularized corneal limbus into the normally avascular cornea.

Hydron pellets, as described in Kenyon et al., Invest Opthamol. & VisualScience 37:1625, 1996, incorporated sucralfate with basic fibroblastgrowth factor (bFGF) (90 ng/pellet), bFGF and IgG (14 μg/pellet,control), or bFGF and TWEAKR-Fc (14 μg). The pellets were surgicallyimplanted into corneal stromal micropockets created by micro-dissection1 mm medial to the lateral corneal limbus of 6-8 week old male C57BLmice. After five days, at the peak of neovascular response to bFGF, thecorneas were photographed, using a Zeiss slit lamp, at an incipientangle of 35-50° from the polar axis in the meridian containing thepellet. Images were digitized and processed by subtractive color filters(Adobe Photoshop 4.0) to delineate established microvessels byhemoglobin content. Image analysis software (Bioquant, Nashville, Tenn.)was used to calculate the fraction of the corneal image that wasvascularized, the vessel density within the vascularized area, and thevessel density within the total cornea.

As shown in Table 1, TWEAKR-Fc (100 pmol) inhibited bFGF (3pmol)-induced corneal angiogenesis, reducing the vascular density to 50%of that induced by FGF alone or FGF+IgG. In addition to reducingvascular area, local administration of TWEAKR-Fc significantly inhibitedFGF induced vessel density (imaged on hemoglobin) by 70% compared to thevessel density in the presence of the control protein IgG-Fc.

TABLE 1 Effect of TWEAKR-Fc on FGF-induced Angiogenesis in the MouseCorneal Pocket Assay Greater than 50% Reduction in Number Treatment andLength of Vessels n/total n (%) FGF alone 0/2 (0%) FGF + IgG 0/2 (0%)FGF + TWEAKR-Fc  6/9 (67%)

Example 5 Qualitative TRAF Binding to the TWEAK Receptor (TWEAKR)Cytoplasmic Domain

Members of the TRAF family are intra-cellular signaling molecules.Several members of the TRAF family are known to associate with membersof the TNF receptor family in order to initiate a signaling cascade thatactivates the NF-kappa-B pathway, resulting in cell activation andproliferation. A qualitative in vitro binding assay was performed totest whether members of the TRAF family of intra-cellular signalingmolecules bind to the cytoplasmic domain of TWEAKR and to learn,therefore, whether the small cytoplasmic domain of TWEAKR is capable ofmediating a signal into the cell via the TRAF pathway.

A GST fusion vector consisting of the C-terminal 29 amino acids ofTWEAKR fused to glutathione S-transferase was created by sub-cloning theappropriate insert into the pGEX-4T (Amersham Pharmacia Biotech) vectorat the BamHI and NotI sites. The product from this vector was expressedin E. coli and bound to sepharose beads as described by Galibert et al.,J. Biol. Chem. 273(51):34120, 1998. Similarly constructed beads coatedwith RANK cytoplasmic domain-GST fusion proteins were used as a positivecontrol, and beads coated with GST alone were used as a negativecontrol. ³⁵S-methionine/cysteine labeled TRAF proteins were produced inreticulocyte lysates (TNT-coupled Reticulocyte Lysate Systems, Promega)according to the manufacturer's protocol. Reticulocyte lysatescontaining the labeled TRAF molecules were first pre-cleared using thecontrol beads followed incubation with the indicated fusion proteincoated beads in binding buffer (50 mM HEPES [pH 7.4], 250 mM NaCl, 0.25%(v/v) Nonidet P-40, 10% glycerol, 2 mM EDTA) at 4 degrees Celsius for 2hours. After washing 4× with binding buffer bound TRAF molecules elutedfrom the beads in SDS-loading buffer, separated by SDS-PAGE, dried andexposed to X-ray film.

Binding above background levels was seen with TRAFS 1, 2 and 3. Nobinding above background levels was seen with TRAFS 4, 5, and 6. Theability of TWEAKR to bind to TRAFs 1, 2, and 3 demonstrates that TWEAKRis capable of inducing a signal to the cell via the TRAF pathway, andtherefore transmitting a proliferative signal into the host cell. Thisexperiment provides further evidence that TWEAKR is the functionalreceptor for TWEAK. It also illustrates a further means by whichsignaling can be inhibited: by disrupting the TRAF-TWEAKR interactionwith a small molecule, or by use of a dominant negative variant of theTRAF molecule.

Example 6 Activity of TWEAKR-Fc in an Endothelial Cell ProliferationAssay

An endothelial cell proliferation assay was used to quantitate theinhibition of bFGF or TWEAK induced-proliferation by TWEAKR-Fc in vitro.In this assay, endothelial cell proliferation is measured after 4 daysof cell growth in microtiter wells using a cell-labeling molecule calledcalcein AM. Esterases expressed by the cells cleave the calcein andcause it to fluoresce when excited at 485 nm. Uncleaved calcein does notfluoresce. The amount of fluorescence is directly related to the numberof endothelial cells in the culture well. Endothelial cell proliferationis often regulated by agents that stimulate and/or inhibit angiogenesisin vivo.

Primary HUVEC (human umbilical vein endothelial cells) were obtainedfrom a commercial source (Clonetics, Walkersville, Md.), cultured, andused at passage 2 to 7. Replicate cultures were set up by adding 3000HUVEC to each microtiter well in endothelial cell basal media (EBM, anendothelial cell basal media that contains no growth factors or serumand is based on the media formulations developed by Dr. Richard Ham atthe University of Colorado, Clonetics) plus 0.05% FBS (fetal bovineserum). At the time of culture initiation FGF-2 (fibroblast growthfactor-2, 10 ng/ml) or human TWEAK (100 ng/ml) was added to the culturesin the presence of human IgG (huIgG, control) or human TWEAKR-Fc atconcentrations ranging from 0.08 μg/ml to 20 μg/ml (0.25 to 20 μg/ml forTWEAK-induced and 0.08 to 6.7 μg/ml for FGF-2-induced). The HUVECcontaining cultures were incubated for 4 days at 37° C., 5% CO₂. On thefourth day of culture 4 μM calcein-AM was added to the cultures and 2hours later the wells were evaluated for fluorescence. The results,expressed as the average fluorescence (485-530 nm) counts for replicatewells plus or minus the SEM, are shown in FIGS. 4 and 5.

TWEAKR-Fc specifically inhibited TWEAK-induced HUVEC proliferation in adose-dependent manner when compared to huIgG, which did not effectTWEAK-induced proliferation (FIG. 4). In addition, TWEAKR-Fc inhibitedthe basal proliferation of HUVEC observed during culture in EBM plus0.05% FBS, as compared to huIgG, which did not. Interestingly, TWEAKR-Fcalso inhibited FGF-2 mediated HUVEC proliferation at concentrations ofgreater than 2 μg/ml, as compared to huIgG, which did not effect theFGF-2 induced HUVEC proliferative response (FIG. 5). These results showthat TWEAKR-Fc inhibits HUVEC proliferation induced by the addition ofexogenous recombinant human TWEAK. That TWEAKR-Fc partially inhibitsserum-induced HUVEC-proliferation indicates HUVEC produce endogenousTWEAK that promotes growth/survival of the EC (endothelial cell) via theTWEAKR. TWEAKR-Fc attenuation of FGF-2 induced proliferation indicatesthat at least part of the EC response to FGF-2 is dependent onendogenous TWEAK/TWEAKR interaction.

In another set of experiments to examine the effects of TWEAKR onproliferation of HUVEC cells a construct was made that fused a syntheticFLAG octapeptide epitope onto the N-terminal extracellular domain ofTWEAKR (FLAG-TWEAKR). The resulting protein was expressed by transienttransfection in HUVEC and incubated with cross-linked anti-FLAGmonoclonal antibody. Cross-linking the receptor in this manner avoidsbackground from the endogenous TWEAKR expressed by HUVEC. Proliferationwas measured by BrdU uptake. Lipid mediated transfection of HUVEC withFLAG-TWEAKR resulted in expression of recombinant FLAG-TWEAKR on thecell surface by 36 hours post transfection. In vitro culture ofFLAG-TWEAKR expressing HUVEC with the complex of M2 anti-FLAG and goatanti-mouse IgG increased BrdU incorporation 3-fold over the level ofBrdU incorporation observed by culturing FLAG-TWEAKR expression cellswith goat anti-mouse IgG alone. Cultures of FLAG-TWEAKR expressing HUVECwith the complex of M2 anti-FLAG and goat anti-IgG increased BrdUincorporation 6-fold over the level of BrdU incorporation observed byculturing vector-only transfected HUVEC with the cross-linking complex.Incubation with the cross-linking complex did not alter BrdUincorporation in vector alone transfected HUVEC. As an additionalcontrol, cells transfected with the FLAG construct that were not exposedto anti-FLAG also showed decreased BrdU uptake relative those that wereexposed to crosslinked anti-FLAG. This data provides additional evidencethat despite the small size of TWEAKR, TWEAKR is capable of initiating aproliferative signal in human endothelial cells.

Example 7 Inhibition of Neovascularization by TWEAKR-Fc in a MurineTransplant Model

Survival of heterotopically transplanted cardiac tissue from one mousedonor to the ear skin of another genetically similar mouse requiresadequate neovascularization by the transplanted heart and thesurrounding tissue, to promote survival and energy for cardiac musclefunction. Inadequate vasculature at the site of transplant causesexcessive ischemia to the heart, tissue damage, and failure of thetissue to engraft. Agents that antagonize the angiopoietins andendothelial specific factors involved in endothelial cell migration andvessel formation can decrease angiogenesis at the site of transplant,thereby limiting graft tissue function and ultimately engraftmentitself.

The following studies were carried out, utilizing a murine heterotopiccardiac isograft model, in order to demonstrate the antagonistic effectsof TWEAKR-Fc on neovascularization. In all experiments, female BALB/c(≈12 weeks of age) recipients received neonatal heart grafts from donormice of the same strain.

TWEAKR-Fc Dose Titration

In the described experiments, the donor heart tissue was engrafted intothe left ear pinnae of the recipient on day 0 and the mice were dividedinto treatment groups. The control group received human IgG (Hu IgG, 400μg/day) while the other treatment groups human TWEAKR-Fc at a dose of400 μg/day or 150 μg/day. All treatments (proteins administered byintraperitoneal injection) began on day 0 and continued for fourconsecutive days. The functionality of the grafts was determined bymonitoring visible pulsatile activity on days 7 and 12 post-engraftment.Table 2 shows the experimental results.

TABLE 2 Functional Heart Isoengraftment Following Dose Titration withTweakR/Fc Treatment Day 7 Day 12 N = Hu IgG 100* 100 3 400 μg HuTWEAKR-Fc 100  100 5 150 μg Hu TWEAKR-Fc 40 80 5 400 μg *all results arereported as percent of mice with pulsatile heart grafts

Administration of TWEAKR-Fc to isograft-bearing mice caused asignificant, dose-dependent, delay in cardiac isoengraftment. Sixtypercent of TWEAKR-Fc-treated mice at the 400 ug/day dose, failed toexhibit pulsatile activity on day 7 post transplant as compared to huIgGcontrol, where no effect on isoengraftment was observed. At this dose,TWEAKkR-Fc administration caused permanent engraftment failure in onefifth of the mice compared to huIgG control where no effect onengraftment was observed. While a dose of 400 μg of huTWEAKR-Fc showed asignificant anti-angiogenic effect, a 150 ug dose of TWEAKR-Fc did notshow measurable activity in this model.

Example 8 Regulation of TWEAKR mRNA Expression I Vascular Smooth MuscleCells (SMC)

Rat aortic SMC were serum-starved and then treated with FGF for variouslengths of time. RNA was isolated and TWEAKR mRNA levels were examinedby Northern Blot hybridization. A single TWEAKR transcript of ≈1.2 kb insize was detected in SMC. TWEAKR mRNA expression was transiently inducedfollowing FGF addition, with maximal levels detected at 2 hours poststimulation. Serum-starved SMC were also treated for 4 hours withvarious agents (e.g., phorbol ester, polypeptide growth factors, peptidehormones) and then a Northern blot was performed to determine whetherTWEAKR gene expression could be induced by multiple distinct growthpromoters. TWEAKR mRNA levels were significantly elevated following PMA,FBS, PDGF-BB, EGF, FGF or Ang II treatment of rat SMC. TGF-beta1, IGF-1and alpha-thrombin treatment had only a slight stimulatory effect. Theseresults indicate that WEAKR is a growth factor-regulated gene invascular SMC.

Example 9 Chromosome Mapping

The gene corresponding to a TWEAKR polypeptide is mapped using PCR-basedmapping strategies. Initial human chromosomal assignments are made usingTWEAKR-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNAkit from BIOS Laboratories (New Haven, Conn.), following themanufacturer's instructions. More detailed mapping is performed using aGenebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville,Ala.; described in Walter, M A et al., Nature Genetics 7:22-28, 1994).Data from this analysis is then submitted electronically to the MITRadiation Hybrid Mapper (URL:http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following theinstructions contained therein. This analysis yields specific geneticmarker names which, when submitted electronically to the NCBI Genemapbrowser(www-ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search?chr=hum_chr.inf&query),yield the specific chromosome interval.

Example 10 TWEAKR Stability

Ligand blots were generated by running either TweakR-Fc or RP-Fc as acontrol on a standard SDS-PAGE and blotted onto nitrocellulose. Theseparate samples were prepared by with and without the addition ofreducing agent (DTT) and with and without heating at 100° C. to denaturethe proteins. This blot was probed with TWEAK-leucine zipper conditionedsupernatants followed by ¹²⁵I labeled M15 anti-leucine zipper. Theresults showed that all TweakR-FC samples strongly bound TWEAK while theRP-Fc samples did not. This shows that TweakR ligand binding domain willspontaneously re-fold into an active conformation even after beginreduced and boiled in SDS loading buffer.

Example 11 Treatment of Tumors with TWEAKR Antagonists

TWEAKR antagonists, including antibodies and TWEAKR-Fc, are tested inanimal models of solid tumors. The effect of the TWEAKR antagonists isdetermined by measuring tumor frequency and tumor growth.

The relevant disclosures of publications cited herein are specificallyincorporated by reference. The examples presented above are not intendedto be exhaustive or to limit the scope of the invention. The skilledartisan will understand that variations and modifications and variationsare possible in light of the above teachings, and such modifications andvariations are intended to be within the scope of the invention.

1-20. (canceled)
 21. An isolated nucleic acid encoding a monoclonalantibody, wherein said antibody binds to a polypeptide consisting ofresidues 28 to 68 of SEQ ID NO:4, binds to the extracellular domain of ahuman TWEAK receptor consisting of the amino acid sequence of SEQ IDNO:4, and inhibits binding of a TWEAK molecule to said TWEAK receptor.22. The isolated nucleic acid of claim 21, wherein said antibody bindsto the cysteine-rich repeat region of said TWEAK receptor.
 23. Theisolated nucleic acid of claim 21, wherein said antibody inhibitsangiogenesis.
 24. The isolated nucleic acid of claim 21, wherein saidantibody promotes angiogenesis.
 25. The isolated nucleic acid of claim21, wherein said human TWEAK receptor is glycosylated.
 26. The isolatednucleic acid of claim 21 wherein said antibody comprises a radioisotope,a plant-derived toxin, a fungus-derived toxin, a bacterial-derivedtoxin, ricin A, or diphtheria toxin.
 27. The isolated nucleic acid ofclaim 21, wherein said antibody is selected from the group consistingof: a) an intact human antibody; b) a human antibody fragment; c) anintact chimeric antibody; d) a chimeric antibody fragment; e) an intacthumanized antibody; f) a humanized antibody fragment; g) a Fab fragment;h) an Fv fragment; i) an F(ab′)2 fragment; and j) a single chainantibody.
 28. An isolated nucleic acid encoding a polypeptide, whereinsaid polypeptide comprises the monoclonal antibody of claim 21 andinhibits binding of a TWEAK molecule to said TWEAK receptor.
 29. Anisolated cell, wherein said cell comprises the isolated nucleic acid ofclaim
 21. 30. The isolated cell of claim 29, wherein said cell is ahybridoma.
 31. The isolated cell of claim 29, wherein said cell is arecombinant cell.